No complementation between TP53 or RB-1 and v-src in astrocytomas of GFAP-v-src transgenic mice.

Human low-grade astrocytomas frequently recur and progress to states of higher malignancy. During tumor progression TP53 alterations are among the first genetic changes, while derangement of the p16/p14ARF/RB-1 system occurs later. To probe the pathogenetic significance of TP53 and RB-1 alterations, we introduced a v-src transgene driven by glial fibrillary acidic protein (GFAP) regulatory elements (which causes preneoplastic astrocytic lesions and stochastically astrocytomas of varying degrees of malignancy) into TP53+/- or RB-1+/- mice. Hemizygosity for TP53 or RB-1 did not increase the incidence or shorten the latency of astrocytic tumors in GFAP-v-src mice over a period of up to 76 weeks. Single strand conformation analysis of exons 5 to 8 of non-ablated TP53 alleles revealed altered migration patterns in only 3/16 tumors analyzed. Wild-type RB-1 alleles were retained in all RB-1+/-GFAP-v-src mice-derived astrocytic tumors analyzed, and pRb immunostaining revealed protein expression in all tumors. Conversely, the GFAP-v-src transgene did not influence the development of extraneural tumors related to TP53 or RB-1 hemizygosity. Therefore, the present study indicates that neither loss of RB-1 nor of TP53 confer a growth advantage in vivo to preneoplastic astrocytes expressing v-src, and suggests that RB-1 and TP53 belong to one single complementation group along with v-src in this transgenic model of astrocytoma development. The stochastic development of astrocytic tumors in GFAP-v-src, TP53+/- GFAP-v-src, and RB-1+/- GFAP-v-src transgenic mice indicates that additional hitherto unknown genetic lesions of astrocytes contribute to tumorigenesis, whose elucidation may prove important for our understanding of astrocytoma initiation and progression.

Astrócitos imunorreativos à proteína glial fibrilar ácida (GFAP) em sistema nervoso central de equinos normais e de equinos com leucoencefalomalácia

A proteína glial fibrilar ácida (GFAP), subunidade dos filamentos intermediários do citoesqueleto celular, está presente no citoplasma de astrócitos. Técnicas imunohistoquímicas com anticorpos primários anti-GFAP são geralmente empregadas para identificar astrócitos no sistema nervoso, permitindo verificar também sua hipertrofia. Vários estudos mostram a distribuição, a morfologia e a citoarquitetura de astrócitos em várias regiões do SNC do homem e de animais de laboratório. No entanto, em animais domésticos e, especialmente em equinos, poucas informações estão disponíveis. No presente trabalho, verificou-se a densidade e a morfologia de astrócitos imunorreativos à GFAP na substância branca da córtex cerebral de equinos com leucoencefalomalácia (LEM) comparando-se esses aspectos com o de equinos normais. Animais com LEM apresentaram hipertrofia de astrócitos em áreas próximas às lesões, representada pelo aumento do corpo celular, do núcleo e dos prolongamentos citoplasmáticos. O número de astrócitos apresentou-se reduzido e a imunorreatividade foi mais acentuada. Nos animais normais, verificou-se distribuição constante de astrócitos imunorreagentes com características de fibrosos. Alterações vasculares nos animais com LEM, como por exemplo degeneração de endotélio vascular, também foram observadas, podendo estar associadas às alterações astrocíticas.

Control of the phosphorylation of the astrocyte marker glial fibrillary acidic protein (GFAP) in the immature rat hippocampus by glutamate and calcium ions: possible key factor in astrocytic plasticity

The present review describes recent research on the regulation by glutamate and Ca2+ of the phosphorylation state of the intermediate filament protein of the astrocytic cytoskeleton, glial fibrillary acidic protein (GFAP), in immature hippocampal slices. The results of this research are discussed against a background of modern knowledge of the functional importance of astrocytes in the brain and of the structure and dynamic properties of intermediate filament proteins. Astrocytes are now recognized as partners with neurons in many aspects of brain function with important roles in neural plasticity. Site-specific phosphorylation of intermediate filament proteins, including GFAP, has been shown to regulate the dynamic equilibrium between the polymerized and depolymerized state of the filaments and to play a fundamental role in mitosis. Glutamate was found to increase the phosphorylation state of GFAP in hippocampal slices from rats in the post-natal age range of 12-16 days in a reaction that was dependent on external Ca2+. The lack of external Ca2+ in the absence of glutamate also increased GFAP phosphorylation to the same extent. These effects of glutamate and Ca2+ were absent in adult hippocampal slices, where the phosphorylation of GFAP was completely Ca2+-dependent. Studies using specific agonists of glutamate receptors showed that the glutamate response was mediated by a G protein-linked group II metabotropic glutamate receptor (mGluR). Since group II mGluRs do not act by liberating Ca2+ from internal stores, it is proposed that activation of the receptor by glutamate inhibits Ca2+ entry into the astrocytes and consequently down-regulates a Ca2+-dependent dephosphorylation cascade regulating the phosphorylation state of GFAP. The functional significance of these results may be related to the narrow developmental window when the glutamate response is present. In the rat brain this window corresponds to the period of massive synaptogenesis during which astrocytes are known to proliferate. Possibly, glutamate liberated from developing synapses during this period may signal an increase in the phosphorylation

Glial fibrillary acidic protein (GFAP): modulation by growth factors and its implication in astrocyte differentiation

Intermediate filament (IF) proteins constitute an extremely large multigene family of developmentally and tissue-regulated cytoskeleton proteins abundant in most vertebrate cell types. Astrocyte precursors of the CNS usually express vimentin as the major IF. Astrocyte maturation is followed by a switch between vimentin and glial fibrillary acidic protein (GFAP) expression, with the latter being recognized as an astrocyte maturation marker. Levels of GFAP are regulated under developmental and pathological conditions. Upregulation of GFAP expression is one of the main characteristics of the astrocytic reaction commonly observed after CNS lesion. In this way, studies on GFAP regulation have been shown to be useful to understand not only brain physiology but also neurological disease. Modulators of GFAP expression include several hormones such as thyroid hormone, glucocorticoids and several growth factors such as FGF, CNTF and TGFß, among others. Studies of the GFAP gene have already identified several putative growth factor binding domains in its promoter region. Data obtained from transgenic and knockout mice have provided new insights into IF protein functions. This review highlights the most recent studies on the regulation of IF function by growth factors and hormones.

Papel das reservas intracelulares de cálcio no efeito de aminoácidos excitatórios sobre a fosforilação da proteína ácida fibrilar glial (GFAP) em hipocampo de ratos jovens

A proteína ácida fibrilar glial (GFAP) é uma proteína da classe dos filamentos intermediários, exclusivamente expressa em astrócitos no sistema nervoso central (SNC). A função específica da fosforilação desta proteína é ainda desconhecida. No entanto, tem sido demonstrado que o equilíbrio dinâmico entre o estado fosforilado e desfosforilado de sítios específicos da GFAP pode regular a polimerização e despolimerização dos filamentos intermediários durante eventos de estruturação do citoesqueleto glial. Nosso grupo de pesquisa demonstrou que a fosforilação da GFAP em hipocampo de ratos jovens (P12-P16) é estimulada no mesmo nível por glutamato, via um receptor glutamatérgico metabotrópico do grupo II (mGluR II), e pela ausência de Ca2+ externo (presença de EGTA). Entretanto, o tratamento simultâneo com glutamato e EGTA não resulta em efeito sinergístico, sugerindo um mesmo mecanismo de ação para estas duas situações estimulatórias da fosforilação da GFAP (WofchuK & Rodnight, 1994; Kommers et al., 1999; Rodnight et al., 1997). Este mecanismo provavelmente não envolve reservas intracelulares de Ca2+ associadas a receptores de IP3, uma vez que mGluRs II estão envolvidos com o mecanismo de transdução de sinal via adenilato ciclase e não via hidrólise de fosfoinositídios. Uma hipótese proposta é de que o glutamato, via mGluR, bloqueia canais de Ca2+ tipo L, inibindo uma cascata de desfosforilação dependente de Ca2+, associada a GFAP (Rodnight et al., 1997). Interessantemente, os receptores rianodina (RyRs) presentes nas reservas intracelulares de Ca2+ reguladas por tais receptores estão associados com canais de Ca2+ tipo L (Chavis et al., 1996). Com base nestes dados, buscou-se neste trabalho avaliar se a modulação glutamatérgica da fosforilação da GFAP em fatias de hipocampo de ratos jovens envolve as reservas intracelulares de Ca2+ reguladas por RyRs e se o Ca2+ proveniente destas reservas atua de maneira semelhante ao Ca2+ oriundo do espaço extracelular. Nossos resultados mostraram que há uma evidente participação do Ca2+ proveniente das reservas intracelulares reguladas por RyRs no mecanismo modulatório da fosforilação da GFAP via ativação de mGluRs em fatias de hipocampo de ratos jovens, uma vez que a cafeína e a rianodina (agonistas de RyRs) revertem totalmente o efeito estimulatório do agonista glutamatérgico metabotrópico 1S,3R-ACPD sobre a fosforilação da proteína e este efeito da cafeína é inibido por dantrolene (antagonista de RyRs). Talvez o Ca2+ oriundo das reservas reguladas por RyRs tenha o mesmo papel do Ca2+ proveniente do espaço extracelular, ou seja, desencadeia uma cascata de desfosforilação associada à GFAP mediada pela calcineurina, uma vez que quelando o Ca2+ intracelular livre com BAPTA-AM, após a mobilização destas reservas, tal efeito não ocorre. A participação de receptores adenosina (AdoRs) e do AMP cíclico (AMPc) ainda permanece a ser estudada. Entretanto, é sabido que em ratos jovens a ativação de mGluRs aumenta a formação de AMPc potenciando o efeito de outros tipos de receptores, como os AdoRs e, provavelmente, isto é mediado por um mGluR II (Schoepp & Johnson, 1993; Winder & Conn, 1996). Neste trabalho mostrou-se justamente o possível envolvimento de tais mecanismos de transdução de sinal na modulação da fosforilação da GFAP, pois a adenosina deaminase (enzima que metaboliza adenosina endógena) e a forscolina (agente que estimula a enzima adenilato ciclase) alteraram o nível de fosforilação da GFAP. Estes resultados evidenciam o envolvimento das reservas intracelulares de Ca2+ reguladas por RyRs no mecanismo de transdução de sinal que modula o estado de fosforilação GFAP mediado pela ativação de mGluRs.

Astrócitos imunorreativos à proteína glial fibrilar ácida (GFAP) em sistema nervoso central de equinos normais e de equinos com leucoencefalomalácia

A proteína glial fibrilar ácida (GFAP), subunidade dos filamentos intermediários do citoesqueleto celular, está presente no citoplasma de astrócitos. Técnicas imunohistoquímicas com anticorpos primários anti-GFAP são geralmente empregadas para identificar astrócitos no sistema nervoso, permitindo verificar também sua hipertrofia. Vários estudos mostram a distribuição, a morfologia e a citoarquitetura de astrócitos em várias regiões do SNC do homem e de animais de laboratório. No entanto, em animais domésticos e, especialmente em equinos, poucas informações estão disponíveis. No presente trabalho, verificou-se a densidade e a morfologia de astrócitos imunorreativos à GFAP na substância branca da córtex cerebral de equinos com leucoencefalomalácia (LEM) comparando-se esses aspectos com o de equinos normais. Animais com LEM apresentaram hipertrofia de astrócitos em áreas próximas às lesões, representada pelo aumento do corpo celular, do núcleo e dos prolongamentos citoplasmáticos. O número de astrócitos apresentou-se reduzido e a imunorreatividade foi mais acentuada. Nos animais normais, verificou-se distribuição constante de astrócitos imunorreagentes com características de fibrosos. Alterações vasculares nos animais com LEM, como por exemplo degeneração de endotélio vascular, também foram observadas, podendo estar associadas às alterações astrocíticas.

Aberrant E-cadherin, beta-catenin, and glial fibrillary acidic protein (GFAP) expression in canine choroid plexus tumors

Expression of E-cadherin and beta-catenin has been widely studied in various human and canine epithelial tumors and has been correlated with dedifferentiation, invasiveness, and metastasis. Choroid plexus tumors (CPTs) are of epithelial origin, and the most important prognostic factor in human medicine is the tumor grade. Limited information is available regarding E-cadherin and beta-catenin expression in human CPTs, and no information is found in the veterinary literature. In the current study, 42 canine CPTs (19 choroid plexus papillomas and 23 choroid plexus carcinomas) were retrospectively reviewed, and the intensity and cellular staining pattern of E-cadherin and beta-catenin were correlated with histological features, paying special attention to grade, invasion, and metastasis. In addition, cytokeratin and glial fibrillary acidic protein (GFAP) antibodies were evaluated as markers for canine CPTs. It was found that loss of E-cadherin and beta-catenin expression was uncommon in canine CPTs. Rather, membranous expression of both molecules was increased in CPTs compared to normal choroid plexus (NCP), regardless of tumor grade. Additionally, aberrant cytoplasmic or nuclear expression of both E-cadherin and beta-catenin was often observed in CPTs. GFAP was frequently expressed in CPTs in contrast to NCP. None of these parameters were correlated with malignancy, and therefore, do not appear to be useful for prognostic information. Nevertheless, a panel of antibodies including E-cadherin and GFAP might be useful to support the diagnosis of CPTs and help to differentiate them from other tumors, such as ependymomas and metastatic epithelial tumors.

Targeted deletion in astrocyte intermediate filament (Gfap) alters neuronal physiology.

Glial fibrillary acidic protein (GFAP) is a member of the family of intermediate filament structural proteins and is found predominantly in astrocytes of the central nervous system (CNS). To assess the function of GFAP, we created GFAP-null mice using gene targeting in embryonic stem cells. The GFAP-null mice have normal development and fertility, and show no gross alterations in behavior or CNS morphology. Astrocytes are present in the CNS of the mutant mice, but contain a severely reduced number of intermediate filaments. Since astrocyte processes contact synapses and may modulate synaptic function, we examined whether the GFAP-null mice were altered in long-term potentiation in the CA1 region of the hippocampus. The GFAP-null mice displayed enhanced long-term potentiation of both population spike amplitude and excitatory post-synaptic potential slope compared to control mice. These data suggest that GFAP is important for astrocyte-neuronal interactions, and that astrocyte processes play a vital role in modulating synaptic efficacy in the CNS. These mice therefore represent a direct demonstration that a primary defect in astrocytes influences neuronal physiology.

GFAP analysis in aluminium chloride-treated rat brain regions

The neurotoxicity of aluminium chloride was assessed in maleWistar albino rats. Rats were treated with aluminium chloride dissolved in distilled water at a dose of 300 mg/kg body weight daily by oral gavage (1 ml) for 45 days. Controls were treated with distilled water only. Animals were sacrificed and different brain regions were dissected. GFAP analysis was carried out by Western blotting using mouse anti-GFAP monoclonal antibody (Pharmingen: 60311D) at 1:1000. Blots were developed with HRP-linked goat anti-mouse secondary antibody and quantified by densitometry.

Role Of Astrocytes In Thiamine Deficiency.

Thiamine deficiency (TD) is the underlying cause of Wernicke's encephalopathy (WE), an acute neurological disorder characterized by structural damage to key periventricular structures in the brain. Increasing evidence suggests these focal histological lesions may be representative of a gliopathy in which astrocyte-related changes are a major feature of the disorder. These changes include a loss of the glutamate transporters GLT-1 and GLAST concomitant with elevated interstitial glutamate levels, lowered brain pH associated with increased lactate production, decreased levels of GFAP, reduction in the levels of glutamine synthetase, swelling, alterations in levels of aquaporin-4, and disruption of the blood-brain barrier. This review focusses on how these manifestations contribute to the pathophysiology of TD and possibly WE.